Ceramide kinase-like proteins

ABSTRACT

A ceramide kinase like protein encoded by a polynucleotide of SEQ ID NO: 1 to SEQ ID NO: 9 and its use.

The present invention relates to organic compounds, such as a protein,e.g. a ceramide kinase like protein.

Sphingolipids have been considered as one of the major components of thecell membrane. Recent evidence has shown that, beyond their structuralrole, they can act as bioactive lipids and impact on signaltransduction, in a way that is reminiscent of what is occurring withglycerophospholipids.

Physiological activity of sphingolipid metabolites include e.g.induction of apoptosis and stimulation of cell proliferation and it hasbeen suggested that enzymes which metabolise sphingolipids are expectedto participate in the induction of various diseases.

For example

-   -   ceramide which controls cell mechanisms has been suggested to be        a regulator in the enzymatic reaction indicated above, see e.g.        Hannun, Y. A. et al, TIBS 20, 73-77 (1995);    -   Spiegel, S. et al, Curr. Opin. Cell. Biol. 8, 159-167 (1996) and        Mathias S. et al, Science 259, 619-622 (1993) have reported that        ceramide works as a second messenger of inflammatory cytokines,        such as TNF-α and IL-1β, and activates arachidonic pathways,        such as phospholipase A₂; and ceramide thus may be considered as        an exacerbating factor in inflammatory disorders;    -   ceramide exacerbates the reduction of CD4⁺ T-cell accompanied by        apoptosis and HIV infection of brain cells in patients infected        with HIV, see e.g. Coline M. G. et al, Proc. Assoc. Am.        Physicians 109, 146-153 (1997) and Wilt S. et al, Ann. Neurol.        37, 381-394 (1995);    -   in Begum N. et al, Eur. J. Biochem, 238, 214-220 (1996) and in        Hotamsligil, G. S. et al, Science, 271, 665-668 (1996) it is        reported that TNF-α may cause insulin restistance in type 2        diabetes mellitus as a trigger and obesity and that ceramide is        involved in the downregulation of TNF-α;    -   in Beuttler B. et al, J. Cardiovasc. Pharmacol. 25, S1-S8 (1995)        it is disclosed that ceramide triggers septicemia caused by        lipopolysaccharide;    -   according to Schissel, S. L. et al in J. Clin. Invest.,        1455-1464 (1996) the increase of ceramide activates        sphingomyelinase in the aggregating reaction of LDL which        triggers atherosclerosis lesions;    -   from Michael J. M. et al, Cancer Res., 57, 3600-3605 (1997),        Bose R. et al, Cell, 82, 405-414 (1995), and Jaffrezou, J. P. et        al, EMBO J., 15, 2417-2424 (1996) it is known that ceramide        promotes apoptosis of cancer cells in radiotherapy and        chemotherapy;    -   by Itoh M. et al, Clinical Cancer Res. 8,415-23 (2003) it is        shown that ceramide regulation is involved in drug resistance of        leukemia cells: a decrease of ceramide level is associated with        the chemoresistant condition in leukemia.

Also ceramide-1-phosphate (Cer-1-P), which is produced from ceramide bythe action of ceramide kinase, e.g. by phosphorylation of the hydroxylgroup at position 1 of various ceramide derivatives, e.g. includingN-acylated-, such as N-hexanoyl-, N-octanoyl-,N-palmitoyl-D-erythro-sphingosine, shows physiological activities, e.g.

-   -   Cer-1-P produced by ceramide kinase upon calcium stimulation        regulates the release of neuronal transmitters from brain        synapses, see e.g. Bajjalieh S. M. et al, J. Biol. Chem. 264,        14354-14360 (1989) and modulating the action of ceramide kinase        is thus expected to be of value in the treatment of various        neuronal disorders, e.g. including Alzheimer's disease;    -   Cer-1-P is believed to inhibit various normal ceramide        activities, see e.g. Dressler K. A. et al, J. Biol. Chem. 265,        14917-14921 (1990), maybe through inhibition of acid        sphingomyelinase as evidenced by Gomez-Munoz A. et al, J. Lipid        Res. 45, 99-105 and thus Cer-1-P is expected to modulate, e.g.        suppress, various disorders, e.g. inflammatory disorders, e.g.        including chronic arthritis, HIV-infection, type 2 diabetes        mellitus caused by insulin resistance as a trigger, obesity,        septicemia and atherosclerosis; i.e. by activation of ceramide        kinase it is believed that such diseases may be treated;    -   Cer-1-P is believed to act primarily inside the cell where it        facilitates vesicle transport. It has been implicated in        phagocytosis, and therefore could play an important role during        inflammation processes, Hinkovska-Galcheva V. T. et al, J. Biol.        Chem 273, 33203-9 (1998);    -   the mitogenic activity of exogenously added Cer-1-P has also        been shown (Gomez-Munoz A. et al Biochem J 325, 435-40 (1997).        Therefore, this sphingolipid metabolite may be relevant to cell        proliferation disorders, including but not limited to cancer and        psoriasis.    -   Cer-1-P has been reported to mediate cytokine-and calcium        ionophore-induced arachidonic release, by Pettus, B. J. et        al, J. Biol. Chem. 278, 38206-13 (2003), and this group further        indicated that C-1-P may directly activate cytosolic PLA2        (Pettus, B. J. et al, J. Biol. Chem 279, 11320-6, 2004) and this        further evidences the possible role of Cer-1-P in inflammatory        disorders;    -   Cer-1-P levels could also be relevant to the pathophysiology        (e.g. susceptibility to retinitis pigmentosa) of the visual        system as suggested by Tuson M. et al, Am. J. Hum. Genet.        74:128-38 (2004).

We have now found a polynucleotide (gene) which encodes a ceramidekinase like protein.

In one aspect the present invention provides an isolated polynucleotideof sequence SEQ ID NO:1.

We also have found a polynucleotide of sequence SEQ ID NO:2 and apolynucleotide of sequence SEQ ID NO:3 which constitute 5′, or 3′,respectively, untranslated regions of a polynucleotide of sequence SEQID NO:1. The sum of polynucleotides of the sequences SEQ ID NO:1, SEQ IDNO:2 and SEQ ID NO:3 provides a 3203 bp cDNA of polynucleotide sequenceSEQ ID NO:4.

In another aspect the present invention provides an isolatedpolynucleotide of sequence

-   -   SEQ ID NO:2,    -   SEQ ID NO:3, or    -   SEQ ID NO:4.

We also have found variants of a polynucleotide of sequence SEQ ID NO:1,e.g. of polynucleotide sequences SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8 and SEQ ID NO:9, which we have found to be splice variantsof a poylnucleotide of sequence SEQ ID NO:1.

In another aspect the present invention provides an isolatedpolynucleotide which is a splice variant of a polynucleotide of sequenceSEQ ID NO:1, such as an isolated polynucleotide of sequence

-   -   SEQ ID NO:5,    -   SEQ ID NO:6,    -   SEQ ID NO:7,    -   SEQ ID NO:8, or    -   SEQ ID NO:9.

Isolated polynucleotide means that the polynucleotide is isolated fromits natural environment.

We have found that the gene or polynucleotide of sequence SEQ ID NO:1provides the open reading frame (ORF) for encoding a ceramide kinaselike protein, e.g. a splice variant of a polynucleotide of sequence SEQID NO:1 encodes a part of such protein. We have further estimated that aceramide kinase like protein according to the present invention has thefunction of a ceramide kinase.

In another aspect the present invention provides a ceramide kinase likeprotein encoded by a polynucleotide of sequence

-   -   SEQ ID NO:1,    -   SEQ ID NO:5,    -   SEQ ID NO:6,    -   SEQ ID NO:7,    -   SEQ ID NO:8, or    -   SEQ ID NO:9,        e.g. a ceramide kinase like protein encoded by a polynucleotide        of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ        ID NO:9, each is a part of a ceramide kinase like protein        encoded by a polynucleotide of sequence SEQ ID NO:1.

We have found that a polynucleotide of sequence

-   -   SEQ ID NO:1 encodes a polypeptide of sequence SEQ ID NO:10,    -   SEQ ID NO:5 encodes a polypeptide of sequence SEQ ID NO:11,    -   SEQ ID NO:6 encodes a polypeptide of sequence SEQ ID NO:12,    -   SEQ ID NO:7 encodes a polypeptide of sequence SEQ ID NO:13,    -   SEQ ID NO:8 encodes a polypeptide of sequence SEQ ID NO:14, and    -   SEQ ID NO:9 encodes a polypeptide of sequence SEQ ID NO:15.

In another aspect the present invention provides an isolated polypeptideof sequence

-   -   SEQ ID NO:10,    -   SEQ ID NO:11,    -   SEQ ID NO:12,    -   SEQ ID NO:13,    -   SEQ ID NO:14, or    -   SEQ ID NO:15.

A polynucleotide of the present invention is designated herein as “geneof (according to) the present invention”. A ceramide kinase like proteinencoded by a gene of the present invention, which could phosphorylateceramide itself or a metabolite of ceramide, is designated herein as“ceramide kinase like protein of (according to) the present invention”.

A gene of the present invention includes a nucleic acid sequence of SEQID NO:1, e.g. and allelic variants therof, and their complements; andsplice variants thereof, e.g. such as polynucleotides of sequences SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, e.g.including a polynucleotide that hybridizes to a nucleic acid sequence ofa gene of the present invention, e.g. under stringent conditions. E. g.the nucleotide sequence of a gene of the present invention includes asequence, which is different, e.g. as a result of the redundancy(degeneracy) of the genetic code, from the sequence of a gene of thepresent invention, but also encodes a ceramide kinase like protein ofthe present invention, e.g. a part thereof, e.g. having the samebiological activity e.g. or encodes a ceramide kinase like protein ofthe present invention having an amino acid sequence which has at least80%, e.g. 80% to 100%, such as 90%, e.g. 95%, e.g. 97%, e.g. 99% or100%, identity; said identity being calculated by n_(a)=x_(a)−(x_(a).y), wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in said corresponding amino acid sequence,and y is percent identitiy divided by 100 and having the same biologicalactivity; e.g. including a ceramide kinase like protein of the presentinvention produced with an allelic variant or a splice variant of a geneof the present invention. “Identity” is a measure of the identity ofnucleotide sequences or amino acid sequences and may e.g. be calculatedby techniques according to a method as conventional, e.g. by, e.g.commercially available, computer programs. “Stringent conditions”includes that hybridization will occur only if there is at least 80%,e.g. 90%, such as 95%, 97% or 99% identity between the nucleotidesequence of a gene of the present invention and the correspondingpolynucleotide that hybridizes.

A ceramide kinase like protein of the present invention includes apolypeptide with an amino acid sequence encoded by a gene of the presentinvention, e.g. of SEQ ID NO:10, e.g. including a part of a ceramidekinase like protein, such as of polypeptides of sequence SEQ ID NO:11,SEQ ID NO:12 SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, and including apolypeptide with an amino acid sequence which has at least 80% identitywith an amino acid sequence of a polypeptide of the present invention.

A ceramide kinase like protein of the present invention may be in theform of the “mature” polypeptide, or may be part of a largerpolypeptide, such as a fusion protein; e.g. it may be advantageous toinclude an additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purificationsuch as multiple histidine residues, or an additional sequence forstability during recombinant production into a polypeptide of a ceramidekinase like protein of the present invention.

A ceramide kinase like protein of the present invention also includes apolypeptide fragment of a polypeptide of a ceramide kinase like proteinof the present invention, e.g. such as encoded by a splice variant of apolynucleotide of the present invention. Such polypeptide fragment ismeant to be a polypeptide having an amino acid sequence that entirely isthe same in part, but not in all, of the amino acid sequence of apolypeptide of a ceramide kinase like protein of the present invention.Such polypeptide fragment may be “free-standing,” or may be part of alarger polypeptide of which such polypeptide fragment form a part orregion, most preferably as a single continuous region. Preferably suchpolypeptide fragment retains the biological activity of thecorresponding ceramide kinase of the present invention, at least inpart.

A polypeptide of a ceramide kinase like protein of the present inventionalso includes variants of defined polypeptide (fragment) sequences of aceramide kinase like protein of the present invention. Preferredvariants are those that vary from the referents by conservative aminoacid substitutions, e.g. variants wherein a residue is substituted byanother of similar characteristics in the biological function.

“Polynucleotide”, if not otherwise specified herein, includes anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA, or modified RNA or DNA, including without limitation singleand double stranded RNA, and RNA that is a mixture of single- anddouble-stranded regions. “Polypeptide”, if not otherwise specifiedherein, includes any peptide or protein comprising amino acids joined toeach other by peptide bonds. Isolated polypeptide means that thepolypeptide is isolated from its natural environment.

A ceramide kinase like protein of the present invention may be obtainedanalogously to a method as conventional, e.g. using standard cloningfrom a cDNA library derived from mRNA of brain cells comprising a geneof the present invention.

The polynucleotide encoding the ceramide kinase like protein of thepresent invention is obtained from a brain cDNA library. We havedesigned primers to cover the full putative open reading frame (ORF)identified in silico, and have used said primers in a Polymerase ChainReaction protocol in order to amplify the ORF. We have obtained afragment with the expected size and we performed cloning and sequencingwhich confirmed the expected sequence and further enabled us to detectvarious splice variants. By sequence comparison with a known ceramidekinase (accession number AB079066), we conclude that the ceramide kinaselike protein of the present invention is a novel member of a CeramideKinase subfamily of the Diacylglycerol Kinase domain containing enzymesuperfamily, e.g. because of homology considerations.

A gene of the present invention may be used for the recombinantproduction of a ceramide kinase like protein of the present invention.The polynucleotide of SEQ ID NO:1 includes the open reading frame (ORF)for encoding the sequence for the mature ceramide kinase like protein ofthe present invention, e.g. and optionally may contain other codingsequences, such as those encoding a leader or secretory sequence, a pre-or pro- or prepro- protein sequence, or other fusion peptide portions.

For example, a marker sequence which may facilitate purification of afused polypeptide comprising a ceramide kinase like protein of thepresent invention can be encoded. The marker sequence may be anappropriate marker sequence, e.g. including conventional markersequences, e.g. a hexa-histidine peptide, as provided in the pQE vector(Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA(1989)86:821-824, or an HA tag. Any polynucleotide according to thepresent invention may also contain non-coding 5′ and 3′ sequences (suchas a polynucleotide of sequence SEQ ID NO:4), such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

A polynucleotide of the present invention may be introduced into avector which may be used for genetically engineering (transforming,transfecting) a host cell, intended to express a polypeptide of thepresent invention.

In another aspect the present invention provides a vector comprising apolynucleotide of the present invention.

A vector comprising a gene of the present invention may be produced asappropriate, e.g. according to a method as conventional. An appropriatevector may be provided as appropriate, e.g. according to a method asconventional. A vector comprising a gene of the present invention may beuseful to obtain an expression system which is able to produce aceramide kinase like protein of the present invention encoded by a geneof the present invention recombinantly, e.g. in a host cell, such as ina compatible host cell. E. g. for recombinant production of a ceramidekinase like protein of the present invention a host cell may begenetically engineered, e.g. by use of a vector comprising a gene of thepresent invention, to incorporate into the host cell an expressionsystem, e.g. or a part thereof, for expressing a ceramide kinase likeprotein of the present invention. Cell-free translation systems may alsobe used to produce a gene of the present invention, e.g. using RNAsderived from an DNA construct comprising a polynucleotide according tothe present invention, e.g. analogously to a method as conventional.

In another aspect the present invention provides an expression systemcomprising a, e.g. pre-isolated, polynucleotide, e.g. DNA or RNA, of thepresent invention, wherein said expression system or part thereof iscapable of producing a ceramide kinase like protein of the presentinvention when said expression system or part thereof is present in acompatible host cell.

In another aspect the present invention provides

-   -   a host cell comprising an expression system according to the        present invention;    -   a process for producing a ceramide kinase like protein according        to the present invention comprising culturing an isolated host        cell comprising an expression system according to the present        invention under conditions sufficient for the production of a        ceramide kinase like protein of the present invention in the        culture, and recovering, e.g. isolating, said ceramide kinase        like protein of the present invention from the culture;    -   a process for the production of a recombinant host cell which        produces a ceramide kinase like protein according to the present        invention comprising transforming or transfecting a host cell        with the expression system according to the present invention        such that the host cell, under appropriate culture conditions,        produces a ceramide kinase like protein according to the present        invention; and    -   a recombinant host cell produced by transforming or transfecting        a host cell with the expression system according to the present        invention such that the host cell, under appropriate culture        conditions, produces a ceramide kinase like protein according to        the present invention.

Introduction of polynucleotides into host cells may be effected asappropriate, e.g. analogously to a method as conventional, e.g.according to Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986);Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989) such ascalcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction orinfection. Host cells may be easily found. Examples of appropriate hostcells include e.g. bacterial cells, such as streptococci, staphylococci,E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such asyeast cells and Aspergillus cells; insect cells such as Drosophila S2and Spodoptera Sf9 cells; isolated animal cells such as CHO, COS, HeLa,C127, CCL39, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plantcells.

Appropriate expression systems include e.g. chromosomal, episomal andvirus-derived systems, e.g. vectors derived from bacterial plasmids,from bacteriophage, from transposons, from yeast episomes, frominsertion elements, from yeast chromosomal elements, from viruses suchas baculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, e.g. cosmids andphagemids. An expression systems may contain control regions thatregulate as well as engender expression. Generally, any system or vectorsuitable to maintain, propagate or express polynucleotides to produce apolypeptide in a host may be used. The appropriate nucleotide sequencemay be inserted into an expression system as appropriate, e.g.analogously to a method as conventional, e.g. according to Sambrook etal, MOLECULAR CLONING A LABORATORY MANUAL (supra).

A ceramide kinase like protein according to the present invention may berecovered (isolated) and purified from recombinant cell cultures asappropriate, e.g. analogously to a method as conventional, e.g.including detergent extraction, ultracentrifugation, ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, e.g. high performance liquid chromatography. If aceramide kinase like protein according to the present invention isdenatured during isolation and or purification, regeneration of theactive conformation, e.g. refolding of a denaturated polypeptide, may becarried out as appropriate, e.g. analogously to a method asconventional.

A ceramide kinase like protein of the present invention provides inseveral aspects a target for a pharmaceutical against various diseases,e.g. including neuronal disorders, inflammatory disorders, e.g.psoriasis, HIV-infection, type 2 diabetes mellitus, e.g. caused byinsulin restistance as a trigger, obesity, septicemia atherosclerosis,cancer, e.g. and retinitis pigmentosa. For example, by activation of aceramide kinase it is reasonable that such diseases may be treated.Treatment includes treatment and prophylaxis. A ceramide kinase may alsobe useful in a method for detecting and/or quantifying the amount ofceramide in a sample. A splice variant of a gene of the presentinvention may be useful for screening for a full length ceramide kinaselike protein, e.g. and as a diagnostic reagent. A splice variant such asthe one reported in SEQ ID NO:6 would generate a truncated enzyme,bearing the catalytic domain but missing the C-terminal sequence whichmay present regulatory regions underlying substrate specificity orintracellular localization. Alternatively, such shorter form of anucleotide of the present invention may be more suitable for assaydevelopment. A splice variant, e.g. of SEQ ID NO:7, would be evenshorter and may lack kinase activity, and could act as a dominantnegative mutant.

For example a gene (fragment, such as a splice variant) of the presentinvention or a ceramide kinase like protein (fragment) of the presentinvention may be used as a research reagent and as a tool for thediscovery of treatments and diagnostics to animal and human diseases.

In another aspect the present invention provides the use of apolynucleotide, polypeptide or an antibody of the present invention as adiagnostic reagent.

Detection of a mutated form of a gene of the present inventionassociated with a dysfunction will provide a diagnostic tool, e.g. in adiagnostic assay, that may add to or define a diagnosis of a disease orsusceptibility to a disease which results from under-expression,over-expression or altered expression of the corresponding gene or amutant version therof. Individuals carrying mutations in thecorresponding gene may be detected at the DNA level e.g. analogously toa method as conventional. This may be illustrated e.g. by the splicevariant of SEQ ID NO:6, which bears two stretches of repeated DNAsequences (satellite DNA), flanking an additional exon.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatcally by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in the analysis similarily.Deletions and insertions may be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationsmay be identified by hybridizing amplified DNA to labeled genenucleotide sequences of the present invention. Perfectly matchedsequences may be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in electrophoreticmobility of DNA fragments in gels, with or without denaturing agents, orby direct DNA sequencing, e.g. according to Myers et al, Science (1985)230:1242. Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method, e.g. according to Cotton et al, Proc Natl AcadSci USA (1985) 85: 4397-4401. An array of oligonucleotides probescomprising the gene nucleotide sequence of the present invention orfragments thereof may be constructed to conduct efficient screening ofe.g. genetic mutations. Array technology methods may e.g. be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability, e.g. according toM. Chee et al, Science, Vol 274, pp 610-613, 1996.

A diagnostic assay offers a process for diagnosing or determining asusceptibility to diseases mediated by the action of a ceramide kinaselike protein , e.g. including inflammatory disorders, e.g. chronicarthritis, psoriasis, e.g. including neuronal disorders, HIV-infection,type 2 diabetes mellitus caused by insulin restistance as a trigger,obesity, septicemia, artherosclerosis, cancer, e.g. and retinitispigmentosa. Such diseases may be diagnosed e.g. analogously to a methodas conventional, e.g. by methods comprising determining from a samplederived from a subject an abnormally decreased or increased level of

-   -   a ceramide kinase like protein of the present invention,    -   a secondary metabolite of such ceramide kinase like protein,        such as ceramide-1-phosphate, or a related lipid        metabolite-phosphate,    -   a gene mRNA of the present invention.

Decreased or increased expression levels can be determined at the RNAlevel, e.g. according to a method as conventional for the quantitationof polynucleotides, such as, for example, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods. Assay techniques thatmay be used to determine levels of a protein, such as a ceramide kinaselike protein of the present invention, or to detemine secondarymetabolites of such ceramide kinase like protein in a sample derivedfrom a host may be carried out as appropriate, e.g. analogously to amethod as conventional. Such assay techniques include radioimmunoassays,competitive-binding assays, Western Blot analysis, ELISA and methods fordetecting the amount of secondary metabolites, e.g.ceramide-1-phosphate, e.g. including fluorescent methods, massspectrometry and chromatography.

In another aspect the present invention provides a diagnostic kit for adisease or susceptibility to a disease, such as inflammatory disorders,e.g. chronic arthritis, psoriasis, e.g. including neuronal disorders,HIV-infection, type 2 diabetes mellitus caused by insulin resistance asa trigger, obesity, septicemia, artherosclerosis, cancer, e.g. andretinitis pigmentosa, comprising as a main component

-   a) a gene (polynucleotide) of the present invention, e.g. including    allelic variants thereof, or a fragment thereof; or a splice variant    thereof, or-   b) a nucleotide sequence complementary to that of (a), or-   c) a ceramide kinase like protein of the present invention, or-   d) an antibody against a ceramide kinase like protein of the present    invention, e.g. any such kit, (a), (b), (c) or (d) may comprise a    substantial component, e.g. including    -   an appropriate environment of a sample,    -   appropriate means to determine the effect of any of a), b), c)        or d) in a sample to be tested.

A gene of the present invention may also be useful for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.The mapping of relevant sequences to chromosomes according to thepresent invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome may be correlated with genetic map data. Correspondingdata is e.g. disclosed e.g. in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region may be identified through linkageanalysis (coinheritance of physically adjacent genes). The differencesin the cDNA or genomic sequence between affected and unaffectedindividuals may also be determined. If a mutation is observed in some orall of the affected individuals but not in any normal individuals, thenthe mutation is likely to be the causative agent of the disease.

A ceramide kinase like protein of the present invention, or fragmentthereof, or cells expressing a polypetide according to the presentinvention can also be used as immunogens to produce antibodiesimmunospecific for the ceramide kinase polypeptide of the presentinvention. The term “immunospecific” means that the antibodies havesubstantially greater affinity for said polypeptide than their affinityfor other related polypeptides. Antibodies generated against suchpolypeptide may e.g. be obtained by administering the polypeptide or anepitope-bearing fragment-analogue or cell to an animal, preferably anon-human, using routine protocols. For preparation of monoclonalantibodies, an appropriate technique which provides antibodies, e.g.produced by continuous cell line cultures, may be used, e.g. includingthe hybridoma technique (Kohler, G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al, Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R. Liss, Inc., 1985). Techniques for the production ofsingle chain antibodies (e.g. U.S. Pat. No. 4,946,778) can also beadapted to produce single chain antibodies to a polypeptide according tothe present invention. Also, transgenic mice, or other organismsincluding other mammals, may be used to express humanized antibodies.Antibodies as described above may be used, e.g. in the isolation or inthe identification of a clone expressing a polypeptide according to thepresent invention or for the purification of a polypeptide (fragment)according to the present invention by affinity chromatography.

In another aspect the present invention provides an antibody against aceramide kinase like polypeptide of the present invention.

A ceramide kinase like protein of the present invention may beresponsible for many biological functions, including many pathologies,e.g. such as described above. Accordingly, it is desirous to findcompounds/drugs which either stimulate (agonists)

-   -   a ceramide kinase of the present invention, e.g. to produce        secondary metabolites,    -   the expression of a gene of the present invention, e.g. to        stimulate ceramide kinase expression,        or which reduce or inhibit (antagonists)    -   the action of a ceramide kinase like protein of the present        invention, e.g. to reduce or inhibit the production of secondary        metabolites    -   expression of a gene of the present invention.

A ceramide kinase like protein of the present invention or functionalmimetics thereof, e.g. according to Coligan et al, Current Protocols inImmunology 1 (2):Chapter 5 (1991), may thus be used to assess thebinding of agonists or antagonists to a receptor part of the polypeptideof the ceramide kinase like protein of the present invention, e.g. incells, cell-free preparations, chemical libraries, and natural productmixtures, e.g. in a screening assay. Such agonists and antagonists maybe used in the treatment of diseases, such as inflammatory disorders,e.g. chronic arthritis, psoriasis, e.g. including neuronal disorders,HIV-infection, type 2 diabetes mellitus caused by insulin restistance asa trigger, obesity, septicemia, artherosclerosis, cancer, e.g. andretinitis pigmentosa.

Screening procedures may involve the production of appropriate cells inwhich ceramide kinase is expressed. Appropriate cells include cells e.g.from mammals, yeast, Drosophila. Cells expressing the ceramide kinaselike protein of this invention (or cell membranes containing theexpressed kinase) may be contacted with a candidate compound to observebinding, or stimulation or inhibition of a functional response.

A screening assay may be used to test a binding of a candidate compoundwherein adherence to the cells bearing the receptor may be detected bymeans of a label directly or indirectly associated with the candidatecompound or In an assay involving competition with a labeled competitor.Inhibitors of activation may be assayed in the presence and in theabsence of a known agonist. A screening assay may comprise the steps ofmixing a candidate compound with a solution containing a ceramide kinaselike protein of the present invention to form a mixture, determiningactivity of said ceramide kinase like protein in the mixture, andcomparing the activity of the mixture with the activity of a standard. Agene (cDNA) according to the present invention, a polypeptide of aceramide kinase like protein according to the present invention andantibodies to such polypeptide of the present invention may also be usedto provide a screening assay for detecting the effect of candidatecompounds on the production of said gene (mRNA) and said polypeptide incells. For example, an ELISA may be constructed for determining cellassociated levels of said polypeptide, e.g. using monoclonal andpolyclonal antibodies according to a method as conventional, and thatELISA may be used to discover agents (agonists or antagonists) which mayincrease or inhibit the production or the activity of said polypeptidefrom suitably manipulated cells or tissues. An assay for screening maybe conducted, e.g. according to a method as conventional.

Examples of potential (ant)agonists of a gene of the present inventioninclude antibodies or, in some cases, oligonucleotides or proteinclosely related to the ligand ((ant)agonist bound to a polypeptide ofsaid gene) of said gene, e.g. a fragment of said ligand, or smallmolecules, which bind to the gene product of the present invention butdo not elicit a response, or the product of a mutated form of the geneof the present invention so that the activity of the receptor isprevented.

Examples of potential (ant)agonists according to the present inventioninclude compounds which bind to a polypeptide of a ceramide kinase likeprotein of the present invention, e.g. including oligopeptides,polypeptides, protein, antibodies, mimetics, small molecules, e.g. lowmolecular weight compounds (LMW's).

Thus in another aspect, the present invention provides a screening assayfor identifying an agonist or an antagonist of a polypeptide of aceramide kinase like protein of the present invention which assaycomprises as a main component

-   a) a polypeptide of a ceramide kinase like protein of the present    invention, or-   b) a recombinant cell expressing a polypeptide of a), or-   c) a cell membrane expressing a polypeptide according of a), or-   d) an antibody to a polypeptide of a);-   e.g. and means for a contact with a candidate compound; e.g. and    means for determining the effect of the candidate compound on any of    a), b), c) or d),-   e.g. determining whether in the presence of the candidate compound    there is a decrease or increase in the production and or the    biological activity of a polypeptide of a);-   e.g. by comparison of the activity of any of a), b), c) or d) in the    presence and in the absence of the candidate compound;-   and in another aspect

A method of identfying an agonist or antagonist, e.g. including ligands,receptors, antibodies or LMW's, which increases or decreases theproduction and/or the biological activity of a polypeptide of a ceramidekinase like protein of the present invention, which comprises

-   A) contacting    -   a) a polypeptde of a ceramide kinase like protein of the present        invention, or    -   b) a recombinant cell expressing a polypeptide of a), or    -   c) a cell membrane expressing a polypeptide of a), or    -   d) an antibody to a polypeptide of a)        with a candidate compound,-   B) determining the effect of the candidate compound on any of a),    b), c) or d);    -   e.g. determining whether in the presence of the candidate        compound there is a decrease or increase in the production and        or the biological activity of a ceramide kinase of the present        invention;        -   e.g. by comparison of the activity of any of a), b), c)            or d) in the presence and in the absence of the candidate            compound,-   C) choosing an agonist or antagonist determined in step B),    e,g, choosing an appropriate candidate compound from which an    agonist/antagonist effect is positively determined in step B).

It will be appreciated that in any such screening assay, a), b), c) ord) may comprise a substantial component. A candidate compound includescompound (libraries), from which the effect on any of a), b), c) or d)is unknown but may be expected. Compound (libraries) include compoundswhich are set out above as (ant)agonists to a polypeptide according tothe present invention. An (ant)agonist is a candidate compound fromwhich an effect on any of a), b), c) or d) has been found in a screeningassay or in a method for identifying (ant)agonists as described above.An (ant)agonist may decrease or increase the production and or thebiological activity of a polypeptide according to the present invention.

In another aspect the present invention provides an agonist or anantagonist, preferably an antagonist, of a polypeptide of a ceramidekinase like protein of the present invention, which is characterized inthat said agonist or antagonist can be provided by a method ofidentfying an agonist or antagonist of the present invention.

An (ant)agonist of a polypeptide according to the present invention maybe used in the treatment of diseases e.g. including neuronal disorders,inflammatory disorders, HIV-infection, type 2 diabetes mellitus, e.g.caused by insulin resistance as a trigger, obesity, septicemiaarteriosclerosis, cancer, e.g. and retinitis pigmentosa. An (ant)agonistof a polypeptide according to the present invention may thus be usefulas a pharmaceutical.

In another aspect the present invention provides an agonist or anantagonist of a polypeptide of a ceramide kinase like protein of thepresent invention for use as a pharmaceutical, e.g. in the treatment ofdiseases, such as inflammatory disorders, e.g. chronic arthritis,psoriasis, e.g. including neuronal disorders, HIV-infection, type 2diabetes mellitus caused by insulin restistance as a trigger, obesity,septicemia, artheroscierosis, cancer, e.g. and retinitis pigmentosa

and, in another aspect

A soluble form of a ceramide kinase like protein of the presentinvention for use as a pharmaceutical, e.g. for the treatment ofdiseases wherein an (ant)agonist of the present invention is suitable.

An (ant)antagonist of a ceramide kinase like protein of the presentinvention may be administered in the form of a pharmaceuticalcomposition.

In another aspect the present invention provides

-   -   A pharmaceutical composition comprising an agonist or an        antagonist of the present invention as an active ingredient in        combination with pharmaceutically acceptable excipient(s), which        is characterized in that said antagonist or agonist can be        provided by a method of identfying an agonist or antagonist of        the present invention;    -   A pharmaceutical composition comprising a soluble form of a        ceramide kinase like protein of the present invention as an        active ingredient in combination with pharmaceutically        acceptable excipient(s).

Such pharmaceutical composition may be produced as appropriate, e.g.according, e.g. analogously, to a method as conventional, e.g. by mixingan (ant)agonist provided by the method steps A), B) and C) withexcipients, e.g. and further processing the mixture obtained, to obtaina pharmaceutical composition for appropriate administration.

In a further aspect the present invention provides a method of treatingabnormal conditions mediated by a ceramide kinase like protein,

-   e.g. a method of treatment of inflammatory disorders, e.g. chronic    arthritis, psoriasis, e.g. including neuronal disorders,    HIV-infection, type 2 diabetes mellitus caused by insulin    restistance as a trigger, obesity, septicemia, artheroscierosis,    cancer, e.g. and retinitis pigmentosa,-   comprising administering a therapeutically effective amount of an    agonist or antagonist of the present invention, e.g. which can be    provided by the method steps A), B) or C) as described above, e.g.    in combination with pharmaceutically acceptable excipient(s);-   e.g. or administering a therapeutically amount of a soluble form of    a polypeptide of a ceramide kinase like protein of the present    invention,-   e.g. in combination with pharmaceutically acceptable excipients,    e.g. in the form of a pharmaceutical composition;    to a subject in need of such treatment.

A phamaceutical composition of the present invention may be administeredby any conventional route, for example enterally, e.g. including nasal,buccal, rectal, oral, administration; parenterally, e.g. includingintravenous, intramuscular, subcutanous administration; or topically;e.g. including epicutaneous, intranasal, intratracheal administration,e.g. in form of coated or uncoated tablets, capsules, (injectable)solutions, solid solutions, suspensions, dispersions, solid dispersions;e.g. in the form of ampoules, vials, in the form of creams, gels,pastes, inhaler powder, foams, tinctures, lip sticks, drops, sprays, orin the form of suppositories.

Preferred forms of systemic administration of a pharmaceuticalcomposition of the present invention include injection, typically byintravenous injection. Other injection routes, such as subcutaneous,intramuscular, or intraperitoneal, may be used. Alternative means forsystemic administration include transmucosal and transdermaladministration, e.g. using penetrants such as bile salts or fusidicacids or other detergents. In addition, if properly formulated inenteric or encapsulated formulations, oral administration may also bepossible. Administration of a composition according to the presentinvention may also be topical and/or localized.

For such treatment, the appropriate dosage will, of course, varydepending upon, for example, the chemical nature and the pharmakokineticdata of a compound of the present invention employed, the individualhost, the mode of administration, the nature and severity of theconditions being treated and whether a polypeptide of a ceramide kinaselike protein of the present invention is used as an active ingredient,or an (ant)agonist of the present invention. However, in general, forsatisfactory results in larger mammals, for example humans, an indicateddaily dosage is In the range from about 0.1 mg to about 1500 mg (ca 10μg/kg to 20 mg/kg of body weight). Variations in the needed dosage,however, maybe expected in view of the variety of compounds availableand the differing efficiencies of various routes of administration. Forexample, oral administration would be expected to require higher dosagesthan administration by intravenous injection. Variations in these dosagelevels may be adjusted as appropriate, e.g. according to standardempirical routine for optimization.

Polypeptides used in treatment may also be generated endogenously in asubject, e.g. in need of such treatment, in treatment modalities oftenreferred to as “gene therapy”, e.g. as described above. Thus, forexample, cells from a subject may be engineered ex vivo with apolynucleotide, such as a DNA or RNA, to encode a polypeptide accordingto the present invention, e.g. by use of a retroviral plasmid vector.Engineered cells may be introduced into a subject in need of suchtreatment.

In the following examples all temperatures are in degree Centigrade andare uncorrected.

Abbreviations Used

-   PCR Polymerase Chain reaction-   ORF Open Reading Frame

EXAMPLE 1

Search for a cDNA clone having homology to human ceramide kinase isbased on partial information from two clones of the NCBI database,BC020465 and XM_(—)087153. Their assembly is expected to allow toproduce a polynucleotide in which a putative ORF coding for a CeramideKinase like protein is detected. The sequence is fully recovered fromhuman chromosome 2.

EXAMPLE 2

Homology

Sequence homology is addressed using the BLAST algorithm and the GAP GCGsoftware. The ORF claimed herein was determined to have 45.662% homologyat the nucleotide level, and 33.871% similarity (24.798% homology) atthe amino acid level with ceramide kinase (cf Accession number AB079066,and J Biol Chem, 2002, vol 277, 23294-300). Like ceramide kinase, thedecuced amino acid sequence of the ORF (gene) of the present inventiondisplays a DAGK like domain. Interestingly, a stretch of proteinsequence well conserved in the ceramide kinase of various species andnot found in the sphingosine kinase consensus sequence, is also presentin the protein sequence deduced from the ORF of the present invention.It is therefore reasonably expected that the protein encoded by the ORFof the present invention is “ceramide kinase like protein”.

BLAST searches also indicate that the ORF of the present inventionlikely has orthologs in rat and mouse.

EXAMPLE 3

Cloning

The following oligonucleotides are designed to amplify the ORF: 5′ccagcctgcgactccgccatgccc 3′ and 5′ ttactttggaatcatttcttccatgcttcctcc 3′A 25 μl PCR recation is run on 2,5 μl human brain cDNA (Marathon braincDNA library, Clontech), 400mM of each of the above mentionedoligonucleotides, Advantage 2 PCR polymerase (Clontech) using thefollowing amplification parameters: 94° for 5 minutes, 5 cycles of 94°for 30 seconds, 65° for 30 seconds, 68° 3 minutes for 30 seconds, 25cycles of 94° for 30 seconds, 58° for 30 seconds, 68° for 3 minutes 30seconds, followed by a final extension step at 68° for 5 minutes. Thisis made on a MJ PTC-200 thermal cycler. Results are analyzed on a 1%agarose gel (SeaPlaque GTG agarose, Cambrex). Three discrete bands areobserved in the 1,6-2,4 kb range. They are cut and their DNA contentextracted (MinElute gel extraction kit, Qiagen). DNA is either cloneddirectly in pCR4-TOPO (Invitrogen), after A addition (A-addition kit,Qiagen), or cloned in pENTR/SD/D-TOPO (Invitrogen) after a subsequentPCR designed to add a CACC sequence at the 5′ end of the DNA fragment.This additional PCR is done with the following primers: 5′caccatgccctggaggaggcgcagg 3′ and 5′ ttactttggaatcatttcttccatgcttcctcc 3′according to the next cycling parameters: 94° for 2 minutes, 5 times:94° for 10 seconds, 72° for 4 minutes, 5 times: 94° for 10 seconds, 69°for 4 minutes, 23 times: 94° for 10 seconds, 67° for 4 minutes, followedby a final extension step of 5 minutes at 72°. Amplified DNA is resolvedon 1% agarose gel and further purified as described above.

TOPO cloning reactions are undertaken according to the manufacturer'sinstructions (Invitrogen). 2 μl of each TOPO reaction mixture is used totransform 50 μl TOP 10 E.coli according to the manufacturer'sinstructions (Invitrogen). Recombinant clones are selected onLB+kanamycin. Plasmids are prepared according to Qiagen's miniprepprocedure.

EXAMPLE 4

Expression

Expression survey is performed by PCR using a human RAPID-SCAN GeneExpression panel (OriGene) and the following oligonucleotides: 5′acattgcacattataatggggcatgtacagctggtcg 3′ and 5′gttactttggaatcatttcttccatgcttcctcc 3′ with the PCR program #2 statedabove. Expression is found to be tissue specific with strong amounts inadrenal gland, kidney, testis and lung, moderate amount in fetal liver,fetal brain, brain, spleen, prostate, bone marrow and stomach, littleamount in muscle, thyroid, pancreas and PBL, and no detectable level inskin, small intestine, heart, colon, placenta, saliva, ovary and uterus.Other PCR-based experiments have shown that the Ceramide kinase likemRNA is also expressed in the trachea and the spinal cord. Detection ofradiolabelled polypeptide after in vitro translation of tagged fusionproteins may be done by Western blot.

EXAMPLE 5

Subcellular fractionation

Upon expression of the ceramide kinase like protein with a C-terminalflag tag followed by cell fractionation, the protein may be recoveredfrom the cytosol as well as with the perimembrane fraction and also asTriton X-100 insoluble material.

EXAMPLE 6

Subcellular localization

Using GFP tagged recombinant proteins expressed in COS-1 cells, theceramide kinase like protein is found in the cytoplasm and in thenucleus, where in particular, it could also localize to nucleoli.

EXAMPLE 7

Using recombinant overexpressed protein in COS-1 cells, the ceramidekinase like protein is found to contain phosphate molecules, primarilyon threonine residues.

EXAMPLE 8

Assay of various substrates, e.g. diacylglycerol, sphingosine, C2-, C6-,C8-, C16-, C18-ceramides, do not reveal activity of the ceramide kinaselike protein. Also within COS-1 cells transiently expressing theceramide kinase like protein and pulse labelled with ³²P, no specificphospholipids can be detected. It may well be that the ceramide kinaselike protein of the present invention needs particular conditions todisplay activity such as, but not limited to, the assistance of acofactor.

1. An isolated polynucleotide of sequence SEQ ID No:4.
 2. An isolatedpolynucleotide according to claim 1, which is of sequence SEQ ID NO: 1.3. An isolated polynucleotide according to claim 1, which is of sequenceSEQ ID NO:2 or SEQ ID NO:3.
 4. An isolated polynucleotide according toclaim 1, which is of sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, or SEQ ID NO:9.
 5. An isolated polynucleotide of any of claim 1encoding a ceramide kinase like protein.
 6. An isolated polynucleotide,which is of sequence SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, or SEQ ID NO:15.
 7. An isolated ceramide kinaselike protein encoded by a polynucleotide of claim
 1. 8. A Vectorcomprising a polynucleotide of claim
 1. 9. An expression systemcomprising a polynucleotide of claim 1, wherein said expression systemor part thereof is capable of producing a ceramide kinase like protein,when said expression system or part thereof is present in a compatiblehost cell.
 10. A host cell comprising an expression system according toclaim
 9. 11. An antibody against a ceramide kinase like polypeptideaccording to claim
 6. 12. Use of a polynucleotide of claim 1 as adiagnostic reagent.
 13. A diagnostic kit for a disease or susceptibilityto a disease comprising as a main component a) a polynucleotideaccording to claim 1, b) a nucleotide sequence complementary to that ofa), c) a ceramide kinase like protein, or d) an antibody against aceramide kinase like protein.
 14. A method of identifying an agonist orantagonist which increases or decreases the production and/or thebiological activity of a polypeptide of a ceramide kinase like proteinaccording to claim 6, which method comprises A) contacting a) apolypeptide of a ceramide kinase like protein according to claim 6, b) arecombinant cell expressing a polypeptide of a), c) a cell membraneexpressing apolypeptide of a), or d) an antibody against a polypeptideof a) with a candidate compound, B) determining the effect of thecandidate compound on any of a), b), c) or d); C) choosing an agonist orantagonist determined in step B.
 15. An agonist or an antagonist of apolypeptide of a ceramide kinase like protein according to claim 6,which is characterized in that said agonist or antagonist can beprovided by a method of claim
 14. 16. An agonist or an antagonist ofclaim 15 for use as a pharmaceutical.
 17. A method of treating abnormalconditions mediated by a ceramide kinase like protein comprisingadministering a therapeutically effective amount of an agonist orantagonist of claim 15 to a subject in need to such treatment.